The present invention relates to the field of production and screening of gene libraries, and more particularly to the generation and screening of normalized genomic DNA libraries from mixed populations of microbes and/or other organisms.
There has been increasing demand in the research reagent, diagnostic reagent and chemical process industries for protein-based catalysts possessing novel capabilities. At present, this need is largely addressed using enzymes purified from a variety of cultivated bacteria or fungi. However, because less than 1% of naturally occurring microbes can be grown in pure culture (Amann, 1995), alternative techniques must be developed to exploit the full breadth of microbial diversity for potentially valuable new products.
Virtually all of the commercial enzymes now in use have come from cultured organisms. Most of these organisms are bacteria or fungi. Amann et al. (Amann, 1995) have estimated cultivated microorganisms in the environment as follows:
These data were determined from published information regarding the number of cultivated microorganisms derived from the various habitats indicated.
Other studies have also demonstrated that cultivated organisms comprise only a small fraction of the biomass present in the environment. For example, one group of workers recently reported the collection of water and sediment samples from the xe2x80x9cObsidian Poolxe2x80x9d in Yellowstone National Park (Barns, 1994) where they found cells hybridizing to archaea-specific probes in 55 % of 75 enrichment cultures. Amplification and cloning of 16S rRNA encoding sequences revealed mostly unique sequences with little or no representation of the organisms which had previously been cultured from this pool, suggesting the existence of substantial diversity of archaea with so far unknown morphological, physiological and biochemical features. Another group performed similar studies on the cyanobacterial mat of Octopus Spring in Yellowstone Park and came to the same conclusion; namely, tremendous uncultured diversity exists (Ward, 1990). Giovannoni et al. (1990) and Torsvik et al. (1990a) have reported similar results using bacterioplankton collected in the Sargasso Sea and in soil samples, respectively. These results indicate that the exclusive use of cultured organisms in screening for useful enzymatic or other bioactivities severely limits the sampling of the potential diversity in existence.
Screening of gene libraries from cultured samples has already proven valuable. It has recently been made clear, however, that the use of only cultured organisms for library generation limits access to the diversity of nature. The uncultivated organisms present in the environment, and/or enzymes or other bioactivities derived thereof, may be useful in industrial processes. The cultivation of each organism represented in any given environmental sample would require significant time and effort. It has been estimated that in a rich sample of soil, more than 10,000 different species can be present. It is apparent that attempting to individually cultivate each of these species would be a cumbersome task. Therefore, novel methods of efficiently accessing the diversity present in the environment are highly desirable.
The present invention addresses this need by providing methods to isolate the DNA from a variety of sources, including isolated organisms, consortias of microorganisms, primary enrichments, and environmental samples, to make libraries which have been xe2x80x9cnormalizedxe2x80x9d in their representation of the genome populations in the original samples, and to screen these libraries for enzyme and other bioactivities.
The present invention represents a novel, recombinant approach to generate and screen DNA libraries constructed from mixed microbial populations of cultivated or, preferably, uncultivated (or xe2x80x9cenvironmentalxe2x80x9d) samples. In accordance with the present invention, libraries with equivalent representation of genomes from microbes that can differ vastly in abundance in natural populations are generated and screened. This xe2x80x9cnormalizationxe2x80x9d approach reduces the redundancy of clones from abundant species and increases the representation of clones from rare species. These normalized libraries allow for greater screening efficiency resulting in the isolation of genes encoding novel biological catalysts.
Screening of mixed populations of organisms has been made a rational approach because of the availability of techniques described herein, whereas previously attempts at screening of mixed population were not feasible and were avoided because of the cumbersome procedures required.
Thus, in one aspect the invention provides a process for forming a normalized genomic DNA library from an environmental sample by (a) isolating a genomic DNA population from the environmental sample; (b) at least one of (i) amplifying the copy number of the DNA population so isolated and (ii) recovering a fraction of the isolated genomic DNA having a desired characteristic; and (c) normalizing the representation of various DNAs within the genomic DNA population so as to form a normalized library of genomic DNA from the environmental sample.
In one preferred embodiment of this aspect, the process comprises the step of recovering a fraction of the isolated genomic DNA having a desired characteristic.
In another preferred embodiment of this aspect, the process comprises the step of amplifying the copy number of the DNA population so isolated.
In another preferred embodiment of this aspect, the step of amplifying the genomic DNA precedes the normalizing step. In an alternate preferred embodiment of this aspect, the step of normalizing the genomic DNA precedes the amplifying step.
In another preferred embodiment of this aspect, the process comprises both the steps of (i) amplifying the copy number of the DNA population so isolated and (ii) recovering a fraction of the isolated genomic DNA having a desired characteristic.
Another aspect of the invention provides a normalized genomic DNA library formed from an environmental sample by a process comprising the steps of (a) isolating a genomic DNA population from the environmental sample; (b) at least one of (i) amplifying the copy number of the DNA population so isolated and (ii) recovering a fraction of the isolated genomic DNA having a desired characteristic; and (c) normalizing the representation of various DNAs within the genomic DNA population so as to form a normalized library of genomic DNA from the environmental sample. The various preferred embodiments described with respect to the above method aspect of the invention are likewise applicable with regard to this aspect of the invention.
The invention also provides a process for forming a normalized genomic DNA library from an environmental sample by (a) isolating a genomic DNA population from the environmental sample; (b) at least one of (i) amplifying the copy number of the DNA population so isolated and (ii) recovering a fraction of the isolated genomic DNA having a desired characteristic; and (c) normalizing the representation of various DNAs within the genomic DNA population so as to form a normalized library of genomic DNA from the environmental sample.
Another aspect of the invention provides a normalized genomic DNA library formed from an environmental sample by a process comprising the steps of (a) isolating a genomic DNA population from the environmental sample; (b) at least one of (i) amplifying the copy number of the DNA population so isolated and (ii) recovering a fraction of the isolated genomic DNA having a desired characteristic; and (c) normalizing the representation of various DNAs within the genomic DNA population so as to form a normalized library of genomic DNA from the environmental sample. The various preferred embodiments described with respect to the above method aspect of the invention are likewise applicable with regard to this aspect of the invention.